{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/49708"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/49708","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Cellular reprogramming in eukaryotes","abstract":"Nearly all cellular processes are controlled by a highly complex genetic and epigenetic network. Although much of this complexity is still unknown to us, we have nonetheless begun exploring, perturbing, and creating genetic circuits in cells. To truly understand and control a cell, we have to build it from scratch, but as of today, this remains a remote possibility. As oppose to the bottom-up approach, a top-down approach is what I have undertaken in my dissertation. Regardless of the route, the ultimate aim is the same – to be able to program cells like we do to robots today. Two general approaches will be discussed in this dissertation. In the first half, we will look into reprogramming via the introduction of external network components such as an artificial transcription factor (gene switch) or a pathway. In one example, we report the development of a gene switch that is able to regulate the endogenous VEGF-A expression in mammalian cell. The gene switch is specifically and reversibly controlled by 4,4’-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, a VEGF-A induction ratio of ~200× can be achieved in HEK293 cells at micromolar concentrations of DHB. In another example, we report the development of a system to assemble a multi-gene pathway and subsequently regulate the entire pathway in yeast using an estradiol gene switch. To demonstrate the utility of the system, we assembled the 5-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand dependent, coordinated expression of all 5 genes as well as the tightly-regulated production of zeaxanthin. In the second half, we will look into reprogramming via the modification of existing network components such as knocking-out or modifying specific genes on a cell’s genome. Site-specific genome editing relies on the ability to create double stranded break at the specific locus. The technique used for my works is based on transcription activator-like effector (TALE) nucleases (TALENs), which allows researchers to design the DNA binding sequence through a simple cipher. In one example, we will discuss the development of a liquid phase high-throughput TALE synthesis platform (fairyTALE) capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency. We will also discuss the continued development of the fairyTALE platform to synthesize paired single-plasmid TALENs which opens up the possibility of using TALENs for multiplex genome engineering and genetic screening.","abstract_html":"Nearly all cellular processes are controlled by a highly complex genetic and epigenetic network. Although much of this complexity is still unknown to us, we have nonetheless begun exploring, perturbing, and creating genetic circuits in cells. To truly understand and control a cell, we have to build it from scratch, but as of today, this remains a remote possibility. As oppose to the bottom-up approach, a top-down approach is what I have undertaken in my dissertation. Regardless of the route, the ultimate aim is the same – to be able to program cells like we do to robots today. Two general approaches will be discussed in this dissertation. In the first half, we will look into reprogramming via the introduction of external network components such as an artificial transcription factor (gene switch) or a pathway. In one example, we report the development of a gene switch that is able to regulate the endogenous VEGF-A expression in mammalian cell. The gene switch is specifically and reversibly controlled by 4,4’-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, a VEGF-A induction ratio of ~200× can be achieved in HEK293 cells at micromolar concentrations of DHB. In another example, we report the development of a system to assemble a multi-gene pathway and subsequently regulate the entire pathway in yeast using an estradiol gene switch. To demonstrate the utility of the system, we assembled the 5-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand dependent, coordinated expression of all 5 genes as well as the tightly-regulated production of zeaxanthin. In the second half, we will look into reprogramming via the modification of existing network components such as knocking-out or modifying specific genes on a cell’s genome. Site-specific genome editing relies on the ability to create double stranded break at the specific locus. The technique used for my works is based on transcription activator-like effector (TALE) nucleases (TALENs), which allows researchers to design the DNA binding sequence through a simple cipher. In one example, we will discuss the development of a liquid phase high-throughput TALE synthesis platform (fairyTALE) capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency. We will also discuss the continued development of the fairyTALE platform to synthesize paired single-plasmid TALENs which opens up the possibility of using TALENs for multiplex genome engineering and genetic screening.","abstract_has_math":false,"creators":["Liang, Jing"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemical Engineering","degree_department":null,"school":null,"contributors":["Zhao, Huimin","Kong, Hyun Joon","Harley, Brendan A.","Belmont, Andrew S."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-05-30T17:05:51Z","date_published":"2014-05-30T17:05:51Z","updated_at":"2026-07-22T22:25:38Z","subjects":["gene switch","artificial transcription factor","gene regulation","transcription activator-like (TAL) effector","transcription activator-like effector nucleus (TALEN)","genome editing"],"languages":["en"],"rights":["Copyright 2014 Jing Liang"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/49708","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin","Kong, Hyun Joon","Harley, Brendan A.","Belmont, Andrew S."]},{"key":"dc:creator","label":"Author","values":["Liang, Jing"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-05-30T17:05:51Z","2016-09-22T20:59:19Z","2014-05"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemical Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["gene switch","artificial transcription factor","gene regulation","transcription activator-like (TAL) effector","transcription activator-like effector nucleus (TALEN)","genome editing"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2014 Jing Liang"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/49708"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Nearly all cellular processes are controlled by a highly complex genetic and epigenetic network. Although much of this complexity is still unknown to us, we have nonetheless begun exploring, perturbing, and creating genetic circuits in cells. To truly understand and control a cell, we have to build it from scratch, but as of today, this remains a remote possibility. As oppose to the bottom-up approach, a top-down approach is what I have undertaken in my dissertation. Regardless of the route, the ultimate aim is the same – to be able to program cells like we do to robots today. Two general approaches will be discussed in this dissertation. In the first half, we will look into reprogramming via the introduction of external network components such as an artificial transcription factor (gene switch) or a pathway. In one example, we report the development of a gene switch that is able to regulate the endogenous VEGF-A expression in mammalian cell. The gene switch is specifically and reversibly controlled by 4,4’-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, a VEGF-A induction ratio of ~200× can be achieved in HEK293 cells at micromolar concentrations of DHB. In another example, we report the development of a system to assemble a multi-gene pathway and subsequently regulate the entire pathway in yeast using an estradiol gene switch. To demonstrate the utility of the system, we assembled the 5-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand dependent, coordinated expression of all 5 genes as well as the tightly-regulated production of zeaxanthin. In the second half, we will look into reprogramming via the modification of existing network components such as knocking-out or modifying specific genes on a cell’s genome. Site-specific genome editing relies on the ability to create double stranded break at the specific locus. The technique used for my works is based on transcription activator-like effector (TALE) nucleases (TALENs), which allows researchers to design the DNA binding sequence through a simple cipher. In one example, we will discuss the development of a liquid phase high-throughput TALE synthesis platform (fairyTALE) capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency. 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Site-specific genome editing relies on the ability to create double stranded break at the specific locus. The technique used for my works is based on transcription activator-like effector (TALE) nucleases (TALENs), which allows researchers to design the DNA binding sequence through a simple cipher. In one example, we will discuss the development of a liquid phase high-throughput TALE synthesis platform (fairyTALE) capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency. 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